During this past year, we have been working on several projects which relate to the sequence analysis of mammalian tRNA and tRNA genes. By hybridization and sequence analysis we have determined: 1) That beef mitochondria contain 24 to 26 distinct, mitochondrially encoded tRNAs and have obtained almost the total nucleotide sequence of 20 of these tRNAs. 2) mammalian mitochondrial tRNAs show little sequence similarities with their corresponding cytoplasmic (either eukaryote or prokaryote) counterparts. 3) These tRNAs are smaller, more A-T rich, show more mismatched base pairing, and lack several of the "universal" structural features normally ascribed to eukaryotic and prokaryote tRNAs. 4) Most mitochondrial tRNAs lack the universal loop IV sequence which is reportedly involved in elongator tRNA-ribosomal A site binding. 5) With the limited number of tRNAs and almost all possible codons read, mitochondrial tRNAs invoke a codon discrimination mechanism which entails G-N wobble and/or reading only two out of the three available code letters. During this next year, we will complete the nucleotide sequences of these beef mitochondrial tRNAs, extend these sequence studies to the human mitochondrial tRNAs and study the biosynthesis of these unique tRNAs by developing a system in which we can express their cloned genes in E. coli and mammalian mitochondrial extracts.